Defining the bilin lyase domain: lessons from the extended phytochrome superfamily

Through pattern searches of genomic databases, new members of the growing family of phytochrome-related genes were identified and used to construct a 130-180 amino acid motif that delimits the bilin lyase domain, a subdomain of the extended phytochrome family that is sufficient for covalent attachment of linear tetrapyrroles (bilins). To test this hypothesis, portions of locus sll0821, a novel phytochrome-related gene from Synechocystis sp. PCC6803 that encodes a large protein with two potential bilin binding sites, were amplified, and the recombinant apoproteins were tested for bilin binding and phytochrome photoactivity. Our experiments indicated that both sites of this protein, termed Cph2 for cyanobacterial phytochrome 2, possessed bilin lyase activity, revealing two distinct classes of bilin lyase domains--those whose bilin adducts are red, far-red reversible and a second class whose bilin adducts are nonphotochromic. Spectroscopic analysis of photochromic phycocyanobilin and fluorescent phycoerythrobilin adducts of a 24-kDa fragment of Cph2 definitively established that the motif identified by pattern searches represents a bona fide bilin lyase domain. Site-directed mutagenesis of highly conserved charged residues within bilin lyase domains of nearly all members of the extended phytochrome superfamily has identified a glutamate residue critical for bilin binding.